Cell-based microarrays and methods of use

ABSTRACT

The invention provides methods and compositions for rapid, sensitive, and highly specific detection of antigen-specific interactions between cytolytic T lymphocytes (CTLs) and antigen presenting cells (APCs). The invention also features compositions, including kits, for use in the methods of the invention.

FIELD OF THE INVENTION

The invention generally relates to use of cell-based microarrays in thedetection of cytotolytic T lymphocyte (CTL)-antigen recognition.

BACKGROUND OF THE INVENTION

The CD8⁺ T lymphocyte subset can play an important role inimmunosurveillance against tumorigenesis and virus infection (Pardoll,D. Annu. Rev. Immunol. 21, 807-839 (2003); Wong, P. & Pamer, E. Annu.Rev. Immunol. 21, 29-70 (2003)). Peptides presented by majorhistocompatibility complex (MHC) class I molecules on diseased cells canbe recognized by CD8⁺ T cells (Yewdell, J. W. & Haeryfar, S. M. Annu.Rev. Immunol. 23, 651-682. (2005)). Selectivity of CTL forantigen-expressing cells can be directed toward targeted mortality inanti-virus or cancer therapy, if antigens are known.

A key step in developing T cell therapies is therefore to identifyCTL-recognized antigens selectively expressed by a desired target cell,such as a tumor cell or virus infected cell. Typically, identificationof such antigens has involved screening cDNA libraries as cDNA poolsthrough a lengthy multi-step process in multi-well plates (Van denEynde, B. Lethe, B., Van Pel, A., De Plaen, E. & Boon, T. J. Exp. Med.173, 1373-1384 (1991); Boon, T. & van der Bruggen, P. J. Exp. Med. 183,725-729 (1996); Van der Bruggen, P. et al. Immunol. Rev. 188, 51-64(2002). Antigen-specific CTLs detect the presence of antigen-encodingDNA sequences in each cDNA pool. Hundreds of genes in individual cDNApools compete for expression in antigen presenting cells (APC), causingdifficulty in identifying rarely expressed antigens. In addition, lowthroughput in assessment of CTL cytotoxicity using current methodshampers antigen identification.

Although microarrays have been used successfully to examine geneexpression profiles on a genome-wide scale, exploitation of microarraysfor cell-based functional screens is yet to be fully realized.Previously, T cell responses to recombinant MHC molecules bound withdefined peptide antigens spotted on microarrays have been demonstrated(Soen et al. PLoS Biology 1, E65 (2003); Stone et al. Proc. Natl. Acad.Sci. USA 102, 3744-3749 (2005)). Such approaches are useful in detectingthe presence of T cells that recognize a known antigen. Although randompeptide libraries could be screened in an MHC protein array as anapproach to identify novel T cell antigens, such a screen would not beable to detect antigens generated by natural processing events in liveAPC and identify their encoding cDNAs.

There is a need in the field for methods for sensitive and rapididentification of CTL antigens. The present invention addresses thisneed.

LITERATURE

Literature of interest includes:

US 2002/0006664; US 2003/0228694; US 2003/0228601; US 2003/0203486; andU.S. Pat. No. 6,544,790; US 2003/0211548; US 2005/0019843; US2001/0041347.

Liu et al. Nat. Med. 8(2):185-189 (2002); Pardoll, D. Annu. Rev.Immunol. 21, 807-839 (2003); Wong, P. & Pamer, E. Annu. Rev. Immunol.21, 29-70 (2003); Yewdell, J. W. & Haeryfar, S. M. Annu. Rev. Immunol.23, 651-682. (2005); Barry, M. & Bleackley, R. C. Nat. Rev. Immunol. 2,401-409 (2002); Van den Eynde et al. T. J. Exp. Med. 173, 1373-1384(1991); Boon et al. J. Exp. Med. 183, 725-729 (1996); Van der Bruggen, Pet al. Immunol. Rev. 188, 51-64 (2002); Ziauddin et al. Nature 411,107-110 (2001); Wu et al. Trends Cell. Biol. 12, 485-488 (2002); Liu etal. Methods Mol. Biol. 263, 125-140 (2004); Amstad et al. Biotechniques31, 608-610, 612, 614, passim (2001); Guerder et al. J. Immunol. 155,5167-5174 (1995); Greenfield, A. et al. Nat. Genet. 14, 474-478 (1996);Soen et al. PLoS Biology 1, E65 (2003); Stone et al. Proc. Natl. Acad.Sci. USA 102, 3744-3749 (2005).

SUMMARY OF THE INVENTION

The invention provides methods and compositions for rapid, sensitive,and highly specific detection of antigen-specific interactions betweencytolytic T lymphocytes (CTLs) and antigen presenting cells (APCs). Theinvention also features compositions, including kits, for use in themethods of the invention.

An advantage of the invention is that it provides a highly sensitive,specific method for detection of cytotolytic T lymphocyte (CTL)-antigenrecognition. Using the methods of the invention, detection ofCTL-mediated killing, which is indicative of a specific interactionbetween a CTL and an antigen-presenting cell (APC), at the single-celllevel, e.g., at the level of a single interaction between a single CTLand a single APC present on the array. The invention is alsoadvantageous in this regard as it requires very little startingmaterial, e.g., with respect to the number of reactive CTLs that need tobe in the sample for detection.

In one aspect, the invention features methods for identifying a CTLantigen, comprising contacting a sample comprising a CTL with an arraycomprising a plurality of recombinant antigen-presenting cells (APCs),where recombinant APCs expressing different, known targetpolynucleotides are provided at different, discrete locations on thearray; and detecting the presence or absence of caspase activity in saidrecombinant APCs by detecting the presence of a fluorescent signalgenerated from a fluorogenic caspase substrate present in saidrecombinant APCs, where the presence of a fluorescent signal in arecombinant APC is indicative of an antigen-specific interaction betweenthe CTL and the recombinant APC and indicates the recombinant APCcontains a target polynucleotide encoding an antigen specificallyrecognized by the CTL.

In embodiments related to this aspect, the target polynucleotides encodea tumor antigen, and antigen of an intracellular pathogen, or anautoantigen. Where the antigen is an antigen of an intracellularpathogen, the antigen can be a viral antigen, bacterial antigen, antigenof a parasite, or fungal antigen. In further related embodiments, thefluorogenic caspase substrate is a fluorogenic multi-caspase substrate.In still further related embodiments, the CTL is in a biological sampleobtained from a subject, or is a clone of a naturally-occurring CTL.

In another aspect, the invention features a method for detecting a CTLhaving an antigen specificity indicative of the presence of, or priorexposure of, a subject to a disease, where the method comprisescontacting a biological sample comprising CTLs from a subject with anarray comprising a plurality of recombinant APCs, where recombinant APCsexpressing different, known target polynucleotides are provided atdifferent, discrete locations on the array, and wherein the targetpolynucleotides encode a disease antigen; and detecting the presence orabsence of caspase activity in said recombinant APCs by detecting thepresence of a fluorescent signal generated from a fluorogenic caspasesubstrate present in said recombinant APCs. The presence of afluorescent signal in a recombinant APC is indicative of anantigen-specific CTL-recombinant APC interaction, and indicates thebiological sample contains a CTL that specifically recognizes thedisease antigen, indicating the subject has or previously had thedisease for which the disease antigen is specific.

In embodiments related to this aspect, the target polynucleotides encodea tumor antigen, and antigen of an intracellular pathogen, or anautoantigen. Where the antigen is an antigen of an intracellularpathogen, the antigen can be a viral antigen, bacterial antigen, antigenof a parasite, or fungal antigen. In further related embodiments, thefluorogenic caspase substrate is a fluorogenic multi-caspase substrate.In still further related embodiments, the CTL is in a biological sampleobtained from a subject, or is a clone of a naturally-occurring CTL.

In still another aspect, the invention features methods of screening acandidate CTL antigen, comprising contacting a cytotoxic T lymphocyte(CTL) with an array surface comprising multiple distinct regionscomprising target antigen presenting cells (APCs), wherein the targetAPCs express different candidate CTL antigens encoded by differentrecombinant target polynucleotides, wherein the polynucleotides areintroduced into APCs by plating APCs onto the array surface comprisingthe polynucleotide under appropriate conditions for introduction of thepolynucleotide into the APC, such that an array location in which thepolynucleotide was deposited corresponds to the location of the targetAPC expressing the polynucleotide; and detecting the presence or absenceof CTL-mediated induction of apoptosis in the target APCs by detecting adetectable signal from a fluorogenic caspase substrate, whereingeneration of a detectable signal from a fluorogenic caspase substrateis indicative of an increase in caspase activation, where detection ofcaspase activation in a target APC indicates the target APC expression aCTL antigen.

In embodiments related to this aspect, the target polynucleotides encodea tumor antigen, and antigen of an intracellular pathogen, or anautoantigen. Where the antigen is an antigen of an intracellularpathogen, the antigen can be a viral antigen, bacterial antigen, antigenof a parasite, or fungal antigen. In further related embodiments, thefluorogenic caspase substrate is a fluorogenic multi-caspase substrate.In still further related embodiments, the CTL is in a biological sampleobtained from a subject, or is a clone of a naturally-occurring CTL.

In another aspect, the invention features, a method of screening acandidate agent for activity in modulating antigen-specific interactionbetween a CTL and an APC, contacting a candidate agent and a CTL with anarray comprising a plurality of recombinant antigen-presenting cells(APCs), wherein recombinant APCs expressing different, known targetpolynucleotides are provided at different, discrete locations on thearray, and wherein the target polynucleotides encode a target antigen ofinterest; and detecting the presence or absence of caspase activity insaid recombinant APCs by detecting the presence of a fluorescent signalgenerated from a fluorogenic caspase substrate present in saidrecombinant APCs. An increase or decrease in a fluorescent signal in arecombinant APC relative to a fluorescent signal produced by CTL-APCinteractions in the absence of the candidate agent indicates thecandidate agent modulates antigen-specific CTL-APC interaction.

In embodiments related to this aspect, the target polynucleotides encodea tumor antigen, and antigen of an intracellular pathogen, or anautoantigen. Where the antigen is an antigen of an intracellularpathogen, the antigen can be a viral antigen, bacterial antigen, antigenof a parasite, or fungal antigen. In further related embodiments, thefluorogenic caspase substrate is a fluorogenic multi-caspase substrate.In still further related embodiments, the CTL is in a biological sampleobtained from a subject, or is a clone of a naturally-occurring CTL.

The present invention can be developed into assays or manufactured intokits to be used in clinical laboratories or hospitals, e.g., fordiagnosis of a disease (e.g., cancer, infectious disease, autoimmunedisease, and the like). The assay can also be utilized in thedevelopment and clinical trials of vaccines and therapeutic drugs fortreating diseases (e.g., cancer, infectious disease, autoimmune disease,and the like).

These and other advantages, aspects, and embodiments will be readilyapparent to the ordinarily skilled artisan upon reading the presentspecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing microarray reverse transfection andscreening for CTL reactivity. (1) DNA spotting. Specific cDNAs invectors are spotted in complex with Effectene transfection reagent ingelatin on microarray slides in a specific pattern. (2) Reversetransfection. Engineered D^(b+)B7.1⁺293T cells seeded on DNA microarray,express specific antigens at defined locations on the array. (3)Antigen-specific CTL killing. CTL specifically induce apoptosis inantigen-expressing targets on microarray. (4) Image-based fluorometricdetection. CTL induced apoptosis is detected through affinity labelingof active caspases in apoptotic cells by cell permeable FLICA, andexpanded image.

FIG. 2 shows detection of antigen specific CTL response to reversetransfected APC on microarray. 293T APC expressing D^(b) and B7.1 werereverse-transfected with pcDNA3 vector alone, or vector encoding EGFP,NP or HY peptide. Dashed circles delineate features on the arraycontaining recombinant target APCs. Open circles represent “red” signalassociated with FLICA binding. All other light-colored images on thedark background of the array represent green fluorescent signalindicative of EGFP expression. Imaging of EGFP expression (top panel,“green”) and FLICA binding (center panel, “red”) was obtained byfluorescence scanning at 50 μm resolution.

FIG. 3. Processing of a GFP fusion protein detected by antigen specificCTL response on microarray. 293T APC expressing Db and B7.1 werereverse-transfected with pcDNA3 vector encoding EGFP or an EGFP-HYpeptide fusion protein and apoptosis of APC induced by HY-specific CTLwas detected by FLICA binding. Dashed circles delineate features on thearray containing recombinant target APCs. Light-colored images on thedark background of the array represent green fluorescent signal in thetop panel and red fluorescent signal in the center panels. Open circlesin the bottom panel represent “yellow” signal where the “red” and“green” signals colocalize, which indicates colocalization of GFPexpression and FLICA binding (caspase activity indicative of CTLkilling). The Images were obtained by fluorescence scanning at 5 micronresolution; EGFP expression (“green”) and FLICA binding (“red”). Bottompanel is a merged image of the upper panels.

Definitions

“Antigen presenting cell” or “APC” as used herein refers to a eukaryoticcell that is capable of, or can be modified to be capable of, expressiona target polynucleotide encoding a polypeptide of interest, andprocessing the polypeptide for presentation of antigen to a cytolytic Tlymphocyte (CTL), on class I MHC.

The terms “cytolytic T lymphocyte”, “cytotoxic T lymphocyte”, “cytolyticT cell”, “cytotoxic T cell”, and “CTL” are used interchangeably hereinto refer to an immune cell that, through antigen-specific interactionwith a peptide antigen presented in MHC Class I on the surface of anAPC, induces antigen-specific killing of the APC.

The term “binds specifically” or “specific interaction” or“antigen-specific interaction” or “specifically recognize” are usedsubstantially interchangeably in the context of an antigen-specific CTLs(CD8⁺ T lymphocytes) to refer to the phenomenon of binding, includingtransient binding, of a CTL to a particular peptide presented in a classI MHC molecule on a target cell, but not substantially to a differentpeptide presented in a class I MHC molecule on a target cell. In thiscontext, “antigen-specific CTL-APC interactions” are those capable ofinitiating apoptosis in the target APC in an antigen-specific manner(through specific binding interactions between the CTL and the APC).

“Antigen-specific CTL-APC interactions” detectable using the methods ofthe invention are those capable of initiating apoptosis in the targetAPC, which initiation of apoptosis is detected by activation of an APCcaspase to cleave or otherwise modify a fluorogenic caspase substrate.In some contexts, the specification refers to “antigen-specifickilling”, which is meant to refer to not only to actual death of APCs asa result of antigen-specific CTL-APC interactions, but also to theinitiation of apoptosis as detected by caspase activation.

As used herein, the terms “antigen-specific killing”, “specific lysis”,or “antigen-specific lysis” refer to initiation “specific lysis” refersto the phenomenon that results from initiation of apoptosis in a targetcell (target APC) based on the presentation on its cell surface of apeptide antigen to an antigen-specific CTL that recognizes suchpeptide/MHC complex, which peptide antigen can be derived from aparticular protein, peptide, glycoprotein, glycolipid, or lipoproteinand the like. Specific CTL-APC interactions can be readily distinguishedfrom non-specific CTL-ACP interactions through the use of appropriatecontrols, e.g., a control cell (e.g., a cell of the same cell type or acell of same cell line, which cell may optionally contain a controlvector used to facilitate transfer and expression of targetpolynucleotides, but lacks a target polynucleotide coding sequence). Aswill be readily apparent to the ordinarily skilled artisan from contextof use, “antigen-specific killing”, “specific lysis”, or“antigen-specific lysis” are used interchangeably herein in reference toa CTL-APC interaction is a shorthand, convenient reference whichencompasses initiation of killing as well as actual cell death, althoughthe latter is not required for detection of antigen-specific CTL-APCinteractions using the inventive methods.

As used herein, the term “nucleic acid” and “polynucleotides” are usedinterchangeable herein to refer to deoxyribonucleic acid (DNA), and,where appropriate, ribonucleic acid (RNA), and usually refers to nucleicacid capable of being expressed in a host cell, more particularly anantigen presenting cell following array-based transfection.“Complementary DNA” (or “cDNA”) as used herein includes recombinantnucleic acid which is suitable for expression without the need forsplicing to remove any intronic sequence that may have been present inthe nucleic acid from which the cDNA may have been derived.

It should be noted that where abbreviations are used to refer tonucleotides (also referred to as bases), including abbreviations thatrefer to multiple nucleotides. As used herein, G=guanine, A=adenine,T=thymine, C=cytosine, and U=uracil. In addition, R=a purine nucleotide(A or G); Y=a pyrimidine nucleotide (A or T (U)); S=C or G; W=A or T(U); M=A or C; K=G or T (U); V=A, C or G; and N=any nucleotide (A, T(U), C, or G). Nucleotides can be referred to throughout using lower orupper case letters. It is also understood that nucleotides sequencesprovided for DNA in the specification also represent nucleotidesequences for RNA, where T is substituted by U.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean apolymer composed of deoxyribonucleotides.

The terms “ribonucleic acid” and “RNA” as used herein refer to a polymercomposed of ribonucleotides. Where sequences of a nucleic acid areprovided using nucleotides of a DNA sequence, it is understood that suchsequences encompass complementary DNA sequences and further alsoencompass RNA sequences based on the given DNA sequence or itscomplement, where uracil (U) replaces thymine (T) in the DNA sequence orits complement.

As used herein, the terms “heterologous nucleic acid” and “foreignnucleic acid” refer to a nucleic acid, e.g., DNA or RNA, that does notoccur naturally as part of the genome of a host cell in which it ispresent as a genomic or episomal element, or which is found in alocation or locations in the genome that differs from that in which itoccurs in nature. Heterologous DNA is usually not endogenous to the cellinto which it is introduced, but has been obtained from another cell.Examples of heterologous nucleic acid of particular interest includetest polypeptides, which polypeptides are of interest for generatingtest CTL antigens for presentation on a recombinant APC.

As used herein, the terms “target nucleic acid”, “target polynucleotide”and “target sequence” are used interchangeably herein to refer to anucleic acid encoding a polypeptide of interest (often referred toherein as a “target polypeptide” or “target antigen”), where the targetpolynucleotide is capable of being expressed in a recombinant host cellproduced by array-based transfection, and the expressed polypeptideprocessed for antigen presentation the resulting recombinant APC.

“Disease antigen” as used herein refers to an antigen derived from apolypeptide, which antigen is indicative of the presence of, or priorexposure of, a subject to a disease or condition.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, and as usedherein generally refers to amino acids that are genetically encodable.The term includes fusion proteins, including, but not limited to, fusionproteins with a heterologous amino acid sequence, fusions withheterologous and homologous leader sequences, with or without N-terminalmethionine residues; immunologically tagged proteins; fusion proteinswith detectable fusion partners, e.g., fusion proteins including as afusion partner a fluorescent protein, β-galactosidase, luciferase, etc.;and the like.

As used herein the term “isolated,” when used in the context of anisolated compound, refers to a compound of interest that is in anenvironment different from that in which the compound naturally occurs.“Isolated” is meant to include compounds that are within samples thatare substantially enriched for the compound of interest and/or in whichthe compound of interest is partially or substantially purified. Theterm “isolated” encompasses instances in which the recited material isunaccompanied by at least some of the material with which it is normallyassociated in its natural state, preferably constituting at least about0.5%, more preferably at least about 5% by weight of the total materialin a given sample (e.g., total protein weight or total nucleic acidweight). For example, the term “isolated” with respect to apolynucleotide generally refers to a nucleic acid molecule devoid, inwhole or part, of sequences normally associated with it in nature; or asequence, as it exists in nature, but having heterologous sequences inassociation therewith; or a molecule disassociated from the chromosome.

“Purified” as used herein means that the recited material comprises atleast about 75% by weight of the total material present in a composition(e.g., of total nucleic acid or of total protein), with at least about80% being preferred, and at least about 90% being particularlypreferred. As used herein, the term “substantially pure” refers to acompound that is removed from its natural environment and is at least60% free, preferably 75% free, and most preferably 90% free from othercomponents with which it is naturally associated.

A polynucleotide “derived from” or “specific for” a designated sequence,such as a target sequence of a target polynucleotide, refers to apolynucleotide sequence which comprises a contiguous sequence ofapproximately at least about 6 nucleotides, preferably at least about 8nucleotides, more preferably at least about 10-12 nucleotides, and evenmore preferably at least about 15-20 nucleotides corresponding to, i.e.,identical or complementary to, a region of the designated nucleotidesequence. The derived polynucleotide will not necessarily be derivedphysically from the nucleotide sequence of interest, but may begenerated in any manner, including, but not limited to, chemicalsynthesis, replication, reverse transcription or transcription, which isbased on the information provided by the sequence of bases in theregion(s) from which the polynucleotide is derived or specific for.Polynucleotides that are derived from” or “specific for” a designatedsequence include polynucleotides that are in a sense or an antisenseorientations relative to the original polynucleotide.

By “transfection” is meant introduction of nucleic acid into a hostcell, which nucleic acid may be present in the host cell as an episomalelement or may be integrated into the recipient host cell chromosome.The host cells generated using the reverse transfection methods inconnection with the present invention can be transiently or stablytransfected, and usually preferably are at least stably transfected.

“Recombinant” as used herein to describe a nucleic acid molecule refersto a polynucleotide of genomic, cDNA, mammalian, bacterial, viral,semisynthetic, synthetic or other origin which, by virtue of its origin,manipulation, or both is not associated with all or a portion of thepolynucleotide with which it is associated in nature. The term“recombinant” as used with respect to a protein or polypeptide means apolypeptide produced by expression of a recombinant polynucleotide.

A “control element” refers to a polynucleotide sequence which aids inthe transcription and/or translation of a nucleotide sequence to whichit is linked. The term includes promoters, transcription terminationsequences, upstream regulatory domains, polyadenylation signals,untranslated regions, including 5′-UTRs and 3′-UTRs and whenappropriate, leader sequences and enhancers, which collectively providefor or facilitate the transcription and translation of a coding sequencein a host cell.

A “fluorescent indicator” refers to an indicator that is fluorescent,and a “fluorogenic indicator” refers to an indicator that that whenmodified (e.g. by interaction with its target molecule) alters (e.g.increases or decreases) its fluorescence. A “fluorogenic indicator” or“fluorogenic composition” is an indicator (indicator composition) ofthis invention that produces a fluorescent signal.

The term “fluorescence” is well known in the art. In the context of afluorescent dye, the term refers to a dye that can be excited at onewavelength of light following which it will emit light at anotherwavelength. Excitation generally occurs at a wavelength in the range offrom about 250 to 750-nm. Emitted wavelengths are generally in the rangeof from about 200 nm to about 300 nm, from about 300 nm to about 400 nm,from about 380 nm to about 400 nm, from about 400 nm to about 430 nm,from about 430 nm to about 500 nm, from about 500 nm to about 560 nm,from about 560 nm to about 620 nm, from about 620 nm to about 700 nm,from about 700 nm to about 1.5 μm, from about 1.5 μm to about 20 μm, orfrom about 20 μm to about 1000 μm.

A fluorophore (fluorescent dye) that is “distinguishable” from anotherfluorophore using standard detection methods and devices (e.g.,fluorescence microscopy), refers to the fact that the spectralproperties of the two fluorophores being compared are detectablydifferent from one another, e.g., the emission of a given fluorophorediffers from the emission of a second fluorophore by at least about 10nm to about 15 nm, from about 15 nm to about 20 nm, from about 20 nm toabout 25 nm, from about 25 nm to about 30 nm, from about 30 nm to about35 nm, from about 35 nm to about 40 nm, from about 40 nm to about 45 nm,from about 45 nm to about 50 nm, from about 50 nm to about 55 nm, fromabout 55 nm to about 60 nm, from about 60 nm to about 65 nm, from about65 nm to about 70 nm, from about 70 nm to about 75 nm, from about 75 nmto about 80 nm, from about 80 nm to about 85 nm, from about 85 nm toabout 90 nm, from about 90 nm to about 95 nm, from about 95 nm to about100 nm, from about 100 nm to about 120 nm, from about 120 nm to about140 nm, from about 140 nm to about 160 nm, from about 160 nm to about180 nm, or from about 180 nm to about 200 nm, or more. In the context ofthe present invention, such distinguishable fluorophores may be used todistinguish a fluorophore that provides a detectable signal uponinduction of caspase activity (e.g., as in a fluorogenic caspasesubstrate) from a fluorophore used as a control to verify expression ofa target polynucleotide (e.g., as in a reporter protein, such as GFP).

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from a subject. Where the biological sample is to becontacted with a recombinant APC array according to the invention,“biological sample” generally refers to samples suspected of containingan antigen-specific cytolytic T cell (CTL), which samples, afteroptional processing, can be analyzed in an in vitro assay. Typicalsamples of interest include any source in which CTLs may be found,including but not necessarily limited to, blood, plasma, serum, fecalmatter, urine, saliva, milk, organs (e.g., thymus, lymph node, spleen),biopsies (e.g., thymus, lymph node, and spleen), and secretions of theintestinal and respiratory tracts. In general, biological samples canalso include samples comprising in vitro cell culture constituentsincluding but not limited to conditioned media resulting from the growthof cells and tissues in culture medium, e.g., recombinant cells, andcell components. Biological samples also encompass primary cells, orcells derived therefrom and the progeny thereof. The definition alsoincludes samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents; washed; or enrichmentfor certain cell population, such as lymphocytes, particularly CTLs, andthe like. The term “biological sample” encompasses a clinical sample,and also includes cells in culture (primary cells or clones of suchcells), tissue samples, organs, bone marrow, and the like.

The term “assessing” includes any form of measurement, and includesdetermining if an element is present or not. The terms “determining”,“measuring”, “evaluating”, “assessing” and “assaying” are usedinterchangeably and includes quantitative and qualitativedeterminations. Assessing may be relative or absolute. “Assessing thepresence of” includes determining the amount of something present,and/or determining whether it is present or absent. As used herein, theterms “determining,” “measuring,” and “assessing,” and “assaying” areused interchangeably and include both quantitative and qualitativedeterminations.

“Analytical specificity” as used herein refers to the ability of adetection system to specifically detect a CTL-APC cytotoxic interaction(e.g., a CTL-APC interaction that results in induction of apoptosis inthe APC) and not provide for a significant detectable signal that may beassociated with cells that are not undergoing such a CTL-APC cytotoxicinteraction.

“Analytical sensitivity” in the context of the methods refers to thenumber of CTL-APC cytotoxic events that are measurable using the methodsof the invention, e.g., detection of a positive result can be indicatedwith only a few (e.g., as few as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25,30, 35, etc.) reactive CTLs present in a sample. For example, themethods of the invention are of such as sensitivity that a CTL-APCcytotoxic event can be detected at the single cell level.

The terms “individual,” “host,” “subject,” and “patient,” usedinterchangeably herein, refer to a mammal, including, but not limitedto, murines, simians, humans, mammalian farm animals, mammalian sportanimals, and mammalian pets.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to a“CTL” includes a plurality of such CTLs and reference to “APC” includesreference to one or more APCs and equivalents thereof known to thoseskilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, recombinantDNA techniques and immunology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., FundamentalVirology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.);A. L. Lehninger, Biochemistry (Worth Publishers, Inc., currentaddition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2ndEdition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds.,Academic Press, Inc.); Oligonucleotide Synthesis (N. Gait, ed., 1984); APractical Guide to Molecular Cloning (1984).

The invention will now be described in more detail.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery of methods for the rapid andsensitive detection of antigens recognized by antigen-specific cytolyticT lymphocytes (CTLs), which methods are amenable to high-throughput.

In general, the invention relates to methods for identifying T cellantigens specifically recognized by a CTL by contacting antigen-specificCTLs with an array having on a surface a plurality of recombinant targetAPCs located at a plurality of locations on the array surface, where thetarget APCs express different recombinant antigens, which antigens areprocessed for presentation to the CTL (on Class I MHC). Usually, theplurality of recombinant target APCs on the array express a plurality ofdifferent recombinant antigens, with the recombinant target APCs beingprovided on the array as distinct features at defined locations(addressable locations), thus allowing for correlation withantigen-specific CTL reactivity with a target APC of a feature at adefined location with the target polynucleotide present on the array andexpressed in the target APC at that location.

Antigen-specific CTL-APC-mediated killing is detected by induction ofactivity of caspase upon a fluorogenic caspase substrate, such that anincrease in a fluorescent signal which serves as a marker of inductionof apoptosis of the target APC and thus identity of a CTL antigenencoded by the target polynucleotide expressed in the target APC.

FIG. 1 provides an exemplary schematic of a method of the invention. Inthis example, a target DNA encoding a polypeptide of interest (e.g.,cDNAs in expression vectors) is spotted along with a transfection agent(or “carrier”) on the microarray slides in a specific pattern (FIG. 1,(1)). For example, the DNA can be provided as a complex with Effectene™transfection reagent in gelatin. The DNAs are spotted on the array in aspecific pattern so as to provide distinct features having specificlocations or “addresses”. Each feature thus has a known target DNA andcan be identified by virtue of its “address” on the array.

FIG. 1, reference (2) denotes the reverse transfection step, in whichhost cells which can serve as APCs are seeded on the DNA microarrayunder conditions suitable to provide for introduction of the DNA intothe host cell, thus providing a recombinant target APC. In a specificexample, the host cell is a mammalian cell (e.g., 293T cells) engineeredto express a MHC complex (e.g., D^(b+)B7.1⁺). The resulting arraycontains target APCs (recombinant host cells) that express specificantigens at defined locations on the array. Stated differently, thearray produced has a plurality of features, which features includetarget APCs express and present an antigen of interest in a MHC complex,where the plurality of features have different antigen-expressing targetAPCs.

The target APCs are loaded with a cell permeable, fluorogenic caspasesubstrate, such as fluorochrome-labeled inhibitors of caspases (FLICA).Exemplary FLICAs include derivatives of valyalanylaspartic acidfluoromethyl ketone (z-VAD-FMK), which detects drug-induced apoptosisthrough affinity labeling of active caspases.

The array is contacted with CTLs under conditions suitable forantigen-specific CTL-APC interaction. CTLs that specifically recognizean antigen presented on an APC induce apoptosis in the APC, which inturn results in induction of caspase activity, which in turn results inmodification of the fluorogenic caspase substrate, such as afluorochrome-labeled inhibitors of capases (FLICA), so as to provide adetectable signal in target APCs that are the target of CTL reactivity(illustrated in FIG. 1, (3), and inset). As illustrated in FIG. 1, (4),image-based fluorometric detection is used to identify features on thearray that are associated with the detectable fluorescent signalgenerated by the induction of apoptosis in one or more target APCs inthat feature. Such features are thus “positive” for CTL-mediatedkilling, and thus contain target DNA encoding a CTL antigen.

Surprisingly, the methods of the invention provide sensitive and rapidassays that provide for direct screening of T cells for reactivity to atarget APC at the for identifying antigen-specific CTL-APC interactionsat individual microarray spots at the single cell-to-cell interactionlevel. The assays of the invention can be used in a variety ofapplications, such as identification of antigens recognized by a CTLand, in other embodiments, detection CTLs having a known antigenspecificity in a biological sample.

The ability to express large numbers of cDNAs simultaneously at discreetaddresses on microarrays, combined with detection of CTL-inducedapoptosis of APCs measured by FLICA retention on those arrays, offers anadvanced strategy for high throughput identification of cDNAs encodingMHC class I-restricted antigens recognized by CTLs. Since the CTLmicroarray assay involves detection of active forms of highly conservedcaspase enzymes, the methods of the invention are feasible for severalmammalian species, including humans (where the MHC-expressing APC is arelevant HLA-expressing APC). The methods of the invention, to which theinventors refer to as “CTL Array” technology, will significantlyaccelerate the screening process for identifying tumor or virus specificantigens on a genome-wide scale, thereby expanding the repertoire ofcandidate antigens for clinical vaccine development and immunotherapies.The CTL Array technology of the invention can also be used in diagnosticsettings to facilitate detection of CTLs reactive with, for example, a Tcell antigen of a pathogen (e.g., virus or other intracellularpathogen), an autoimmune disorder, and the like.

The compositions and methods of the invention will now be described inmore detail.

Cell-Based Arrays

The cell-based arrays for use in the invention are generally composed ofan array having adherent target APCs (recombinant host cells) at definedlocations on the array, which target APCs are recombinant cells producedby transfection with target polynucleotide spotted at the definedlocations. The target APCs thus express a recombinant antigen ofinterest encoded by the target polynucleotide. Exemplary materials forthe various components of the array, as well as exemplary methods ofmaking such arrays, are described below in more detail.

Host Cells for Use as Recombinant Target Antigen Presenting Cells (APCs)on Arrays

Any suitable host cell capable of adhering to a defined location on anarray surface, capable of expressing a target polynucleotide encoding apolypeptide, capable of antigen processing, and is capable of, or can bemodified to be capable of, providing for presentation of the processedpolypeptide for presentation of antigen to a cytolytic T lymphocyte(CTL) (on class I MHC) is a suitable host cell for use in the invention.In general, such a suitable host cell is referred to herein as “antigenpresenting cell” or “APC”, which, as noted above, refers to a eukaryoticcell that is capable of, or can be modified to be capable of, expressiona target polynucleotide encoding a polypeptide of interest, andprocessing the polypeptide for presentation of antigen to a cytolytic Tlymphocyte (CTL), on class I MHC.

In one embodiment, the host cells as “professional APCs”, which cellsinclude macrophages and dendritic cells. Methods for isolating suchcells for use as host cells in the methods of the invention are wellknown in the art. For example, antibodies specific for a cell surfacemarker indicative of an APC of interest can be used to facilitateisolation of a specific population of APCs. For example anti-CD11b/cand/or anti-CD34 antibodies can be attached to the surface of a bead(e.g., magnetic bead) to provide for isolation of dendritic cells.

In another embodiment, the host cell is an engineered APC. For example,a eukaryotic cell, usually a mammalian cell (usually of the same speciesas the CTL source, e.g., a human cell where the CTLs are from human) canbe modified to express a desired MHC Class I molecule, as well as anynecessary co-stimulatory molecules, such as B7-1 and B7-2 molecules,which co-stimulatory molecules are recognized by receptors on thesurface of the CTL (e.g., CD28 or CTLA-4, the receptors for B7 on the Tcell surface). Nucleic acids encoding a number of different MHC Class Imolecules, as well as nucleic acids encoding such co-stimulatorymolecules, are well known in the art.

In one embodiment, the host cell used to generate a target APC is a“null” cell that is deficient or lacks detectable alloreactive MHCmolecules on the cell surface (so as to avoid alloreactivity witheffector CTLs) and expresses or is modified to express a desired Class IMHC complex to provide for antigen presentation to the effector CTLs ofinterest. Exemplary null cells useful in the methods of the inventionare known in the art, as at methods and compositions to provide forproduction of recombinant null cells that can serve as APCs useful inthe present invention.

In general, to avoid high background levels of caspase activityinduction and reduce the incidence of false positive results, host cellsfor transfection on the arrays should be selected so as to avoidalloreactivity with the CTLs to be screened. Thus, the host cells to beused as target APCs should generally be selected so as to be MHC-matchedwith the subject from whom the CTLs to be screened are obtained. Methodsfor MHC typing, and methods of selecting or modifying a host cell foruse as an MHC-matched APC are known in the art.

Arrays Having Bound Target Polynucleotide Encoding Antigens of Interestfor Expression in Host Cells on Array

Any suitable method of making arrays having a DNA of interest for use intransfecting cells, and any suitable method of transfecting cells usingsuch arrays can be applied in the practice of the present invention. Forexample, US 2002/0006664; US 2003/0228694; US 2003/0228601; US2003/0203486; and U.S. Pat. No. 6,544,790, which published applicationsand patents are specifically incorporated by reference herein in theirentireties. Exemplary arrays as may be suitable for use in the presentinvention are discussed in more detail below.

Target Polynucleotides Encoding Polypeptides of Interest for AntigenPresentation

Target polynucleotides include any polypeptide-encoding nucleic acidwhich is adapted for expression in a host cell to provide a recombinantAPC for use on the arrays useful in the methods of the invention. Ingeneral, a target polynucleotide can be any nucleic acid encoding apolypeptide of interest, where the target polynucleotide is capable ofbeing expressed in a recombinant host cell produced by array-basedtransfection, and the expressed polypeptide processed for antigenpresentation the resulting recombinant APC.

The target polynucleotide can be, for example, DNA, RNA or modified orhybrid forms thereof, with the proviso such are capable of beingexpressed in a recombinant APC. The target polynucleotide may be fromany of a variety of sources, such as nucleic acid isolated from cells,or that which is recombinantly produced or chemically synthesized.

The target polynucleotide can encode a polypeptide of any length,including a full-length polypeptide, or polypeptide fragment,particularly a fragment that provides a T cell antigen that is presentedin Class I MHC for recognition by a CTL specific for that antigen.

The target polynucleotide can be from any suitable source. For example,the transfection array can include coding sequence from cDNAs or genomicDNA. Where the target polynucleotide is naturally occurring, thosesequences can be isolated from any organism or collections of organisms.In addition to native sequences, the coding sequences can include thosewhich have been mutated relative to the native sequence, e.g., a codingsequence that differs from a naturally occurring sequence by deletion,substitution or addition of one or more residues

Target polynucleotide can also be generally by recombinant or synthetictechniques, the latter being most applicable to relatively short nucleicacids. Target polynucleotides can be generated to have randomizedsequences, with the proviso that at least one open reading frame forexpression of a polypeptide is provided.

The target polynucleotide sequences can be present as part of a largervector, such as an expression vector (e.g., a plasmid or viral-basedvector), although such may not be necessary for introduction andexpression of a target polynucleotide. The target polynucleotide can beintroduced into cells in such a manner that at least a sequence defininga coding sequence is integrated into the genomic DNA and is expressed.Alternatively, the target polynucleotide can remain as anextrachromosomal element (e.g., is maintained episomally).

The target polynucleotide as provided on the array for transfection canbe linear or circular, double stranded or single stranded, and can be ofany size suitable for uptake by the host cell. In certain embodiments,especially where traditional expression vectors are used, the targetpolynucleotide is from about 200 nt to about 10 kb in size, usually fromabout 200 nt to about 5 kb, and more usually from about 200 nt to 2 kb.Target polynucleotide can be provided as part of a larger polynucleotide(e.g., as when provided in an expression vector), and in suchembodiments can be from about 1 kb to about 15 kb, usually from about 5kb to about 8 kb.

It is generally desirable that a recombinant target APC produced usingthe reverse transfection technique described herein will be maintainedin the cells and its progeny, i.e., will be capable of replication withand/or in the host cell. It may be a DNA which is integrated into thehost genome, and thereafter is replicated as a part of the chromosomalDNA, or it may be DNA which replicates autonomously, as in the case of aepisomal element. In the latter case, the vector will generally includean origin of replication which is functional in the host. In the case ofan integrating vector, the vector may include sequences which facilitateintegration, e.g., sequences homologous to host sequences, or encodingintegrases. The use of retroviral long terminal repeats (LTR) oradenoviral inverted terminal repeats (ITR) in the construct of thetransfection array can, for example, facilitate the chromosomalintegration of the construct.

The expression vectors may comprise regulatory elements such as anoperably linked promoter, enhancer(s), and/or other 5′ and/or 3′flanking nontranscribed sequences. The expression vectors can furthercomprise 5′ and/or 3′ untranslated sequences, such as ribosome bindingsites, a poly-adenylation site, splice and transcriptional terminationsequences. Exemplary vectors include, but are not limited to,cytomegalovirus (CMV) promoter-based vectors, MMTV promoter-basedvectors, and SV40 promoter-based vectors.

Certain eukaryotic (including mammalian) expression vectors provide forpropagation of the vector in bacteria (such as in an amplification stepafter recovery from the array). Some of these vectors are modified withsequences from bacterial plasmids, such as pBR322, to facilitatereplication and drug resistance selection in both prokaryotic andeukaryotic cells. The various methods employed in the preparation of theplasmids are well known in the art.

Polypeptides Encoded by Target Polynucleotides

The polypeptides encoded by the target polynucleotides can be anypolypeptide of interest, including polypeptides for which identificationof a CTL epitope (if present) is desired, and polypeptides having aknown CTL epitope. The polypeptides can be derived from anaturally-occurring polypeptide, or can be wholly recombinant orsynthetic, as in randomized polypeptides.

In one embodiment the target polynucleotide encodes a polypeptide of acancerous (neoplastic) cell. Such cancers may be associated with solidor semisolid tumors. Exemplary cancers for which identification of CTLantigen is of interest include, but are not limited to lymphomas (e.g.,Hodgkin's, non-Hodgkin's), leukemia, carcinoma, lymphoma, astrocytoma,sarcoma, glioma, retinoblastoma, melanoma, Wilm's tumor, bladder cancer,breast cancer, colon cancer, hepatocellular cancer, pancreatic cancer,prostate cancer, Lung cancer, liver cancer, stomach cancer, cervicalcancer, testicular cancer, renal cell cancer, brain cancer, and thelike.

In one embodiment, the target polynucleotide encodes a polypeptide of anintracellular pathogen (e.g., virus, bacterium, fungi, parasites, andthe like). “intracellular pathogen” refers to any organism that existswithin a host cell, either in the cytoplasm or within a vacuole, for atleast part of its reproductive or life cycle.

Exemplary viral intracellular pathogens from which targetpolynucleotides encoding a polypeptide of interest may be derivedinclude hepatitis (e.g., HBV, HCV, HDV, hepatitis A), retroviruses(e.g., HIV, HTLV-1, HTLV-II), influenza, smallpox, adenovirus,cytomegalovirus, Epstein-Barr virus, HSV (e.g., HSV1, HSV2, HSV6),varicella-zoster virus, papilloma virus, erythrovirus, polyomaviruses(e.g., BK, JC) measles virus, and rubella virus.

Exemplary bacterial intracellular pathogens from which targetpolynucleotides encoding a polypeptide of interest may be derivedinclude Mycobacteria (e.g., M. tuberculosis, M. leprae), Chlamydia,Salmonella (e.g., S. typhi), Legionella, Brucella, Shigella, Neisseria,Staphylococcus, Listeria, enteropathogenic Escherichia coli (EPEC),enterohaemorrhagic Escherichia coli (EHEC), Yersinia, Brucella,Coxiella, Rickettsia, and the like).

Other exemplary intracellular pathogens include protozoa (e.g.,Taxoplasma), fungi, intracellular parasites (e.g., Plasmodium (e.g., P.vivax, P. falciparum, P. ovale, and P. malariae), Leishmania,Trypanasoma, Toxoplasma), and prions.

Other target polynucleotides of interest are those encoding apolypeptide that is derived from an autoimmune antigen or putativeautoimmune antigen.

In other embodiments, the target polynucleotides encode a library ofpolypeptides, which polypeptides can have randomized sequences. In otherembodiments, the subject array can be made of a library of relatedsequences modified relative to one another to provide for a library ofencoded polypeptides, which polypeptides in turn may be processed toprovide for different Class I antigens. Methods for generating one ormore mutants given a desired cDNA are known in the art.

In general, antigenic peptides produced by antigen processing ofrecombinant polypeptide encoded by the target polynucleotide is of alength compatible with presentation with a Class I MHC complex. Suchantigenic peptides are usually from about 6 to 12 amino acids in length,usually from about 8 to 10 amino acids. In general, such antigenicpeptides that render a target APC susceptible to CTL-mediated killingcontain a T cell epitopes. The epitopic sequences from a number ofantigens are known in the art, and may be incorporated for use inscreening in the assays of the invention. Alternatively, the epitopicsequence may be empirically determined using the methods of theinvention (e.g., by using deletion mutants of a selected polypeptide).

Arrays Having Target Polynucleotides

Any suitable method of making arrays having a DNA of interest for use intransfecting cells, and any suitable method of transfecting cells usingsuch arrays can be applied in the practice of the present invention. Forexample, US 2002/0006664; US 2003/0228694; US 2003/0228601; US2003/0203486; and U.S. Pat. No. 6,544,790, which published applicationsand patents are specifically incorporated by reference herein in theirentireties.

Array Substrate

Any suitable surface which can be used to affix the nucleic acidcontaining mixture to its surface can be used. For example, the surfacecan be glass, plastics (such as polytetrafluoroethylene,polyvinylidenedifluoride, polystyrene, polycarbonate, polypropylene),silicon, metal, (such as gold), membranes (such as nitrocellulose,methylcellulose, PTFE or cellulose), paper, biomaterials (such asprotein, gelatin, agar), tissues (such as skin, endothelial tissue,bone, cartilage), minerals (such as hydroxylapatite, graphite).Additional compounds may be added to the base material of the surface toprovide functionality, with the proviso such do not adversely affect thedetection methods as set out in the present invention.

The substrate may be a porous solid support or non-porous solid support.The surface can have concave or convex regions, patterns of hydrophobicor hydrophilic regions, diffraction gratings, channels (e.g.,microfluidics channels) or the like. The surface can be planar, planarwith raised or sunken elements, fibers (e.g. fiber optic bundles),tubular (both interior or exterior), a 3-dimensional network (such asinterlinking rods, tubes, spheres) or other shapes. Where thetransfection array is provided on the end of a fiber optic system, suchas a fiber optic bundle, changes in caspase activity in the cells on thearray in response to CTLs as detected by the fluorogenic caspasesubstrate can be detected spectrometrically by conductance ortransmittance of light over the spatially defined optic bundle.

The surface can be part of an integrated system. For instance, thesurface can be the bottom of a microtitre dish, a culture dish, aculture chamber. In general, the material of the substrate and geometryof the array will be selected based on criteria that it be useful forautomation of array formation, maintaining the recombinant target APCson the surface, contacting the CTLs with the target APCs, and detectionof induction of apoptosis of the APCs were an antigen-specific CTL-APCinteraction occurs.

Characteristics of DNA on Array

The DNA can be provided on the array in a variety of differentconfigurations. For example, the number of different DNAs on the arraycan vary greatly according to the needs of the assay. For example, asingle array can provide at least 10 different DNAs, usually at least100, 500, 750, 1000, 1250, 1500, or 2000 different DNAs per squarecentimeter, where the different DNAs have discrete sequences.Preferably, where the array substrate is a planar surface, the targetsequences are arrayed in an addressable fashion, such as rows andcolumns.

The DNAs are provided as discrete features on the array surface. Theterm “feature”, as it is used in describing a transfection array, refersto an area of a substrate having a known collection of a targetpolynucleotide sequences encoding an antigen of interest or, where thearray has been used in a cell-based transfection procedure, a collectionof recombinant target APCs that are recombinant for a known collectionof recombinant target sequences. One feature is different than anotherfeature if the target sequences of the different features have differentnucleotide sequences.

Usually the feature defines an area having a homogenous collection oftarget polynucleotide sequences, or homogenous collection of recombinanttarget APCs, such that, for example, the population of recombinanttarget APCs present at any one feature contain the same recombinanttarget polynucleotide sequences. Usually the arrays are designed so asto provide for production of recombinant target APCs containing a singlerecombinant target polynucleotide encoding an antigen of interest, whichantigen may be either a known CTL antigen or a candidate CTL antigen.However, features having a population of different targetpolynucleotides, so as to provide for production of a feature containinga population of recombinant target APCs that contain two or morerecombinant target polynucleotides encoding different antigens are alsocontemplated. In these embodiments, the target polynucleotidepopulations (and thus the recombinant target APC population) present inthe feature may be homogenous or heterogenous, usually homogenous.

In some embodiments, the features on the array contain a heterogenouspopulation of target polynucleotide sequences for production of aheterogenous population of recombinant target APCs, different targetpolynucleotides encoding different antigens of interest. Such featureson the array can be produced by pooling different target sequences, andspotting the pooled target sequences at a defined (addressable)location. In such embodiments, where killing of a target APC is detectedafter contact with an antigen-specific CTL at a feature having aheterogenous target APC population, additional screening may beconducted using arrays having homogenous features representing thedifferent pooled recombinant target APCs from the heterogenous featureto facilitate identification of the target APC(s) in the heterogenousfeature that presented an antigen reactive with the CTL(s) (e.g., thepooled target sequences are split among different features on a secondarray, and again screened with the CTL).

If each feature size is about 100 microns on a side, each array may haveabout 100, 500, 750, 1000, 1250, 1500, or 2000 target sequence addresses(features) in a one square centimeter area. In certain preferredembodiments, the transfection array provides a density of at least 100,usually at least 103 different features per square centimeter (103sequences/cm²), and may have more as the limits of the assay allow(e.g., at least 10⁴ features/cm², 10⁵ features/cm², or 10⁶features/cm²).

In some embodiments it may be desirable to provide multiple differenttarget sequences in each feature, e.g., in order to promoteco-transfection of the host cells with at least two different targetsequences, so as to provide for expression of the gene products encodedby each of the two different target sequences. Co-transfections can beaccomplished by including the two or more target polynucleotides in thesolution spotted on the array surface. Usually, the collection ofdifferent target sequences in one feature should be distinct from otherfeatures of the array, however, it may be desirable to provide forco-transfection of a control target sequence which can act as a markerfor expression and/or transfection.

Production of Arrays and Transfection Of Host Cells to ProduceRecombinant APC Target Cells

The DNA encoding a polypeptide of interest is deposited (e.g., spottedor placed in small defined areas) onto a surface (e.g., a slide or otherflat surface, such as the bottoms of wells in a multi-welled plate) indefined, discrete (distinct) locations and allowed to dry, with theresult that the DNA-containing mixture is affixed to the surface indefined discrete locations. Such locations are referred to herein, forconvenience, as defined locations.

The DNA can be deposited in as many discrete locations as desired, andin any pattern desired. The resulting product is a surface bearing theDNA in defined discrete locations; the identity of the DNA present ineach of the discrete locations (spots) is known/defined. The size of theDNA spots and the density of the DNA spots affixed to the surface can beadjusted depending on the conditions used in the methods. For example,the DNA spots can be from about 100 μm to about 200 μm in diameter,usually about 100 μm to about 150 μm in diameter, and can be affixedfrom about 200 μm to about 500 μm apart on the surface. Spots of suchsize on an array can provide for, for example about 1500-2500 spots perstandard slide array.

In general, the DNA is deposited on the array surface as a mixture witha carrier which facilitates transfection. The carrier can be anysuitable material, such as gelatin, a hydrogel (e.g., polycarboxylicacid, cellulosic polymer, polyvinylpyrrolidone, maleic anhydridepolymer, polyamide, polyvinyl alcohol, or polyethylene oxide), or anappropriate lipid-based transfection reagent (e.g., Effectene™). TheDNA-containing mixture is spotted onto a surface, such as a slide, thusproducing a surface bearing the lipid-DNA mixture in defined locations.The array is allowed to dry to affix the lipid-DNA mixture is affixed tothe array surface.

After drying is complete, host cells to be reverse transfected areplaced on top of the surfaces onto which the DNA-containing mixture hasbeen affixed. Actively growing cells are generally used. The host cells(in an appropriate medium) are plated, generally at a relatively highdensity (such as 1×10⁵/cm²), on the array surface having the affixedDNA-containing mixture. The host cells are cultured in an appropriatemedium, such as Dulbecco's Modified Eagles Medium (DMEM) containing 10%heat-inactivated fetal serum (IFS) with L-glutamine andpenicillin/streptomycin (pen/strep). Other media can be used and theircomponents can be determined based on the type of cells to betransfected.

The resulting arrays, which contain the dried DNA-containing mixture andcells into which the DNA is to be reverse transfected, are maintainedunder conditions appropriate for growth of the cells and entry of DNAinto the cell. Usually about one to two cell cycles are sufficient forreverse transfection to occur, but will vary with the cell type andconditions used.

After sufficient time has elapsed, arrays can be assessed fortransfection and/or expression of the encoded product, if desired. Forexample, the DNA spots can include a DNA encoding a reporter gene, whichreporter gene provide a detectable signal (e.g., fluorescence, such aswith GFP, YFP, and the like). The presence of fluorescence indicatesthat reverse transfection has occurred and the encoded protein has beenexpressed in the defined location(s) which show fluorescence. Thepresence of a signal, detected by the method used, on the slidesindicates that reverse transfection of the DNA into cells and expressionof the encoded product or an effect of the DNA in recipient cells hasoccurred in the defined location(s) at which the signal is detected.Since the identity of the DNA present at each of the defined locationsis known, the identity of the expressed protein is also known.

Fluorogenic Caspase Substrates

Detection of CTL-mediated induction of apoptosis in a target cell on thearray is accomplished using a cell-permeable fluorogenic caspasesubstrate. A “fluorogenic caspase substrate” is a peptide-based compoundthat, upon binding and/or cleavage by an activated caspase, provides fora detectable signal, e.g., by generating a fluorescent cleavage productthat is free in the cell or by transferring a detectable label to thecaspase enzyme with which it interacted. For example, SR-VAD-FMK is asulforhodamine derivative of valylalanylaspartic acid fluoromethylketone (VAD-FMK) which is a potent inhibitor of caspase activity. TheSR-VAD-FMK reagent enters the cell and covalently binds to an activatedcaspase, likely by covalently binding to the reactive cysteine (Cys 285)on the large subunit of a caspase heterodimer. The fluorescent label ofSR-VAD-FMK is thought to be transferred to the active site of theactivated caspase enzyme to provide for detection by fluorescencemicroscopy.

Exemplary fluorogenic caspase substrates are composed of twofluorophores (or a fluorophore and a quencher) covalently linked to apeptide (usually of about 18-amino-acids in length) containing aproteolytic cleavage site for a specific caspase or for multipledifferent caspases. In substrates that are not cleaved or bound to anactivated caspase, fluorescence is quenched due to the formation ofintramolecular excitonic dimers. Upon cleavage of the peptide by thespecific caspase or other specific interaction with the activatedcaspase, the fluorophore-fluorophore interaction is abolished, leadingto an increase in fluorescence that can be detected by fluorescencemicroscopy. Since caspase activation in target APC cells occurs shortlyafter the CTL-target APC interaction, detection of caspase activationwithin intact target APC cells provides an early and biologicallyrelevant measurement of CTL-mediated apoptosis, and thus is indicativeof an antigen-specific CTL-APC interaction.

In general, indicators of caspase activity include any chromophore orfluorophore labeled based caspase substrate including, cyclic or linear,mono, dipeptide, tripeptide and tetra peptide to 8, 12, 16, 20, 30, or31 amino acid residue long peptide substrates having attached one or twochromophores or fluorophores or a combination of chromophores andfluorophores, such that the substrate in the uncleaved state (or when itis not bound to activated caspase) does not provide a detectable signalor provides a first detectable signal that can be readily distinguishedfrom a second detectable signal that is generated following cleavage (orbinding to activated caspase).

The caspase activity indicator can a fluorogenic caspase substrate thatis cleaved by any suitable caspase, with the proviso that activity ofthe caspase is induced upon induction of apoptosis in a target cellfollowing a CTL-APC antigen-specific interaction. Fluorogenic caspasesubstrates containing cleavage sequences for each of caspase 3/7(DEVDase), caspase-9 (LEHDase), caspase-8 (IETDase), and caspase-6(VEIDase) have been described (see, e.g., US 2003/0211548). Furthermore,fluorogenic caspase substrates can be selected so as to be cleavedearlier or later in the caspase activation cascade, as desired. Forexample, since caspase-6 is thought to act downstream of caspase-8 and-9, and in some cases caspase-3, in the caspase activation cascade, itmight be expected that more caspase-positive cells may be detected anddetected more quickly using substrates that are cleaved earlier in theapoptotic pathway.

In one embodiment of particular interest, a fluorogenic caspasesubstrate is used which is capable of detecting activation of multiplecaspase enzymes in the target host cell (i.e., a “fluorogenicmulti-caspase substrate”). For example, fluorogenic caspase substrateshaving the VAD peptide can provide for detection of activity of all ofcaspases 1-9.

In general, caspase activity indicators useful in the invention comprisea protease substrate having a fluorescence resonance energy transfer(FRET) system comprising two fluorophores or a chromophore and afluorophore with the fluorescence of the latter quenched until thesubstrate is cleaved by a protease. Certain preferred indicatorscomprise a homo-double labeled substrate (e.g. a substrate attached tofluorophores of the same species) that form an H-dimer (see, e.g., U.S.Pat. Nos. 5,605,809, 5,714,342, and 6,037,137, and internationalapplications WO9613607 WO 98/37226, and WO/01/18238 and variouscommercial reagents (e.g. PhiPhLlux™ from Oncoimmunin, Inc.). Alsocontemplated are substrates that form a J-dimer that results in adecrease in fluorescence when the substrate is cleaved.

CTLs for Screening

CTLs for use in the methods of the invention can be obtained from anysuitable source. For example, the CTLs may be obtained from a subjectwho has, or is suspected to have, CTLs that are reactive with a targetantigen of interest. Typical samples of interest from such subjectsinclude any source in which CTLs may be found, including but notnecessarily limited to, blood, plasma, serum, fecal matter, urine,saliva, milk, organs (e.g., thymus, lymph node, spleen), biopsies (e.g,thymus, lymph node, and spleen), and secretions of the intestinal andrespiratory tracts.

For example, CTLs are obtained from a subject who has been exposed to,or is suspected to have been exposed to, an intracellular pathogen, suchthat the sample obtained from the subject contains or is suspected tocontain CTLs reactive with an antigen of an intracellular pathogen(e.g., virus, intracellular bacteria, intracellular parasite, and thelike as discussed above).

In another example, CTLs are obtained from a subject who has, or issuspected to have, cancer. Thus, samples obtained from such subjectscontain or are suspected to contain CTLs reactive with tumor antigens.

In another example, CTLs are obtained from a subject who has, or issuspected to have, an autoimmune disorder which is cell-mediated. Thus,samples obtained from such subjects contain or are suspected to containCTLs reactive with self-antigens.

CTLs can be primary cells or can be clones. In general, the number ofCTLs used in the screening methods of the invention is relatively low,with usually about 1×10⁶ cells, about 1.5×10⁶ cells, about 2×10⁶ cells,about 3×10⁶ cells, or about 5×10⁶ cells being sufficient for screeningof a typical array as described in the Examples below.

Methods for isolating CTLS are well known in the art. For example, CTLscan be isolated using anti-CD8 antibodies, which may be bound to asubstrate, such as a magnetic bead.

Screening Method

The methods of the invention typically involve contacting target APCs onthe array with a CTL, and detecting the presence or absence ofantigen-specific CTL-APC interactions by detecting the presence orabsence of an activated caspase in the target APC as indicated by adetectable signal from a fluorogenic caspase substrate. Detection caninvolve detecting CTL-APC interactions at the single cell level (e.g.,utilizing a single cell image based instrument).

In general, the methods of the invention can involve detecting thepresence or absence of antigen-specific CTL-APC interactions using aplurality of target APCs contacted with a sample containing CTLs, whichCTLs may share the same antigen specificity or may have differentantigen specificities. In some embodiments it may be desirable toprovide positive or negative control target cells (e.g., provided at oneor more distinct features of the array) that have a labeldistinguishable from the test target APCs on the array. For example,positive control APCs can be provided that present an antigen that isknown to be recognized by a positive control CTL which is included inthe sample. Similarly negative control APCs can be provided that do notpresent an antigen for CTl recognition.

In a preferred embodiment, target APCs are “loaded” with a fluorogeniccaspase substrate prior to or during incubation with the CTLs. Thetarget cells and CTLs (effector cells) are coincubated for a timesufficient for specific interaction between target cells and CTLs,usually about 1-4 hours, although the exact time may vary (e.g.,according to the host cell used as the target cell, the number of CTLsbeing assayed, and the like) and can be readily determined by routinemethods. After a time sufficient for antigen-specific CTL interactionsto occur, the array is washed, and the presence of absence of afluorescence signal is assessed for each feature on the array (e.g., byfluorescence microscopy).

Detection

Arrays can be scanned to detect antigen-specific CTL-APC interactions bya variety of methods, e.g., simple fluorescence microscopy, scanninglaser microscope, by fluorimetry, a modified ELISA plate reader, etc.For example, a scanning laser microscope may perform a separate scan,using the appropriate excitation line. The digital images generated fromthe scan are then combined for subsequent analysis. For any particulararray feature, the ratio of the fluorescent signal prior to contactingwith CTLS and after incubation with CTLs can be determined.

High-Throughput and Automated of Screening

The methods of the invention can be readily adapted for high-throughputassays, which can involve providing for automated screening to detectantigen-specific CTL-APC-mediated killing, as detected by caspaseactivity, which serves as a marker of induction of apoptosis of thetarget APC. Any or all or nearly all steps of the methods of theinvention can be automated, including production of arrays for use intransfection of cells, contacting CTLs with the APC array, and detectionand analysis of antigen-specific CTL interactions.

For example, methods and devices relating to systems for cell-basedscreening see, e.g., US 2001/0041347; US 2003/0096322; US 2004/0101912;US 2004/0063162; US 2004/0009539; US 2003/0204316; and US 2003/0096322,which describe automated systems for analysis of cells containingfluorescent reporter molecules where the cells are provided in an arrayof locations, where, after contacting the cells with a stimulus, imagesare acquired from the cells, and the acquired images analyzed to provideinformation about the effect of the stimulus upon the cell. In thepresent invention, the image acquired would indicate whether anantigen-specific CTL-APC interaction occurred (e.g., at the single celllevel) within the features at one or more addressable locations on thearray, where an increase in detectable signal associated with caspaseactivity induction indicates an antigen-specific CTL-APC interactionoccurred at that feature, and further indicates the target APC containsa target polynucleotide encoding an antigen recognized by a CTL withwhich the array was contacted.

In general, microarray fluorescence readers, such as those typicallyused for expression microarray analysis, can be used to obtain data fromthe “CTL Arrays” described herein. The software typically associatedwith such microarray fluorescence readers can be applied to acquisitionand analysis of data for threshold establishment (i.e., determination ofa background level of fluorescence), standardization and export to otherapplications as desired for further analysis. Optical scanning using anautomated microscope and a motorized stage can be particularly useful inhigh-throughput applications. Robotic loading of slides onto themicroscopy platform allows a further increase in throughput. APC arrayscan be marked with predetermined geographic locations that allowsidentification of array start and stop points. Automated dataacquisition in all involved channels may be performed (for example, butnot limited to brightfield/phase contrast/DIC/Color, FITC, CY5, CY3,DAPI, PI, UV, etc.). Automated analysis is also of interest, allowingautomated counting of antigen-specific CTL-APC interactions (e.g., perfeature), fluorescence intensity, and the like.

Applications of the Methods of the Invention

As will be readily apparent to the ordinarily skilled artisan uponreading the present disclosure, the methods of the invention find avariety of applications in a variety of settings. Exemplary applicationsinclude, but are not limited to, discovery of CTL antigens, detection ofreactive CTLs in a sample (e.g., in the context of diagnosis), and otheruses. Exemplary applications are described below.

Identification of Antigens Recognized by Antigen-Specific CTLs

In one embodiment, the assays of the invention are used to identify anantigen to which CTLs responded in vivo, where the antigen is derivedfrom, for example, a cancerous cell (e.g., a malignant tumor cell,including a metastasis of a primary tumor), an intracellular pathogen,or an autoantigen. CTLs used in this screen are obtained from a subjecthaving a disease or condition in which production of antigen-specificCTLs is suspected, and for which identification of the corresponding CTLantigen(s) is desired.

This embodiment of the invention can be used to facilitate developmentof vaccines, which can be based upon a single CTL antigen or a cocktailof CTL antigens. This embodiment of the invention can also be used totailor vaccines for a given individual or population of individuals,particularly where the individual or population of individuals may havecharacteristics that might affect an immune response in the individualor population. Populations of individuals can be based upon a variety ofdifferent factors, such as one or more of age (e.g., children, youngadults, adults), race or ethnicity (e.g., Caucasian, Afro-American,Hispanic, European (e.g., western or eastern), Asian, Middle Eastern,African, and the like), and immune status (e.g., immunocompromised,having an autoimmune condition, and the like).

Detection of Antigen-Specific CTLs Using Known T Cell Antigens

In another exemplary embodiment, the methods of the invention are usedin diagnostic assay to detect an antigen-specific CTL in a sampleobtained from a subject suspected of having a disease or condition. Insuch assays, detection of an antigen-specific CTL-APC interactionindicates the subject has elicited a CTL response to an antigenassociated with a particular disease or condition (e.g., cancer,infection by an intracellular pathogen, self-antigen, and the like), andthus is affected with such a disease or condition.

Screening for Agents that Modulate Antigen-Specific CTL-APC Interactions

The present invention further provides methods of identifying agentsthat modulate (i.e., “increase” or “decrease”, or “enhance” or“inhibit”, respectively) antigen-specific CTL-APC interactions, and thusmodulates lysis of a target cell. The methods generally involvecontacting the target APCs, or the CTLs, with a candidate agent prior toor during incubation of the CTLs with the target APCs, and detecting theeffect of the candidate agent upon induction of CTL-mediated apoptosisof the target APC.

A reduction in fluorescence in the target APC as a result of CTLinteraction, compared to in the absence of the agent (e.g., in a controlsample), indicates that the agent inhibits antigen-specific cell lysis.Similarly, an increase in fluorescence in the target APC as a result ofCTL interaction, compared to in the absence of the agent (e.g., in acontrol sample), indicates that the agent enhances antigen-specific celllysis. Suitable control samples in such assays are those that do notcontain the test agent. Suitable control samples in such assays arethose that do not contain the test agent.

Agents of interest modulate antigen-specific CTL-target APC interactionssuch that there is an increase or decrease in signal relative (relativeto in the absence of the agent) of at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, or more, when compared with a suitable control.Agents that increase CTL killing are of interest in applications such astreating cancer, treating intracellular pathogen infections, and thelike. Agents that decrease CTL killing are of interest in applicationsuch as treating cell-mediated autoimmune diseases (e.g.,graft-versus-host disease, and the like).

The terms “test agent,” “candidate agent,” “substance,” and “compound”are used interchangeably herein. Candidate agents encompass numerouschemical classes, typically synthetic, semi-synthetic, ornaturally-occurring inorganic or organic molecules. Candidate agents maybe small organic compounds having a molecular weight of more than 50 andless than about 2,500 daltons. Candidate agents may comprise functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, and may contain at least two of thefunctional chemical groups. The candidate agents may comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Agents further encompassinterfering RNA molecules, antibodies, and the like.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidation, etc. to producestructural analogs.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Thecomponents are added in any order that provides for the requisitebinding. Incubations are performed at any suitable temperature,typically between 4.degree. C. and 40.degree. C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hour willbe sufficient.

As is readily apparent, design of the assays described herein aresubject to a great deal of variation, and many formats are known in theart. The above descriptions are merely provided as guidance and one ofskill in the art can readily modify the described protocols, usingtechniques well known in the art.

Kits

Kits for use in connection with the subject invention are also provided.The above-described assay reagents, including, for example, arrayshaving bound cDNAs for array-based transfection of a desired targetcell, target cells having a desired MHC phenotype, one or morefluorogenic caspase substrates, and the like, can be provided in kits,with suitable instructions and other necessary reagents, in order toconduct the assays as described above. Instructions (e.g., written,tape, VCR, CD-ROM, etc.) for carrying out the assay usually will beincluded in the kit. The kit can also contain, depending on theparticular assay used, other packaged reagents and materials (i.e. washbuffers and the like). Standard assays, such as those described above,can be conducted using these kits.

The instructions are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (e.g., associated with the packaging orsubpackaging), etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g., CD-ROM, diskette, etc, including the samemedium on which the program is presented.

In yet other embodiments, the instructions are not themselves present inthe kit, but means for obtaining the instructions from a remote source,e.g. via the Internet, are provided. An example of this embodiment is akit that includes a web address where the instructions can be viewedfrom or from where the instructions can be downloaded.

Still further, the kit may be one in which the instructions are obtainedare downloaded from a remote source, as in the Internet or world wideweb. Some form of access security or identification protocol may be usedto limit access to those entitled to use the subject invention. As withthe instructions, the means for obtaining the instructions and/orprogramming is generally recorded on a suitable recording medium.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Materials and Methods

The following method and material were used in the Example(s) below.

Mice and cell culture. Experiments utilizing mice, 6 to 8 week oldC57BL/6N (B6) strain (Charles River), were approved and followedregulations of the Health Sciences Animal Policy and Welfare Committee,Faculty of Medicine, University of Alberta. The human kidney embryoniccell line 293T (GenHunter Corporation) was grown in DMEM supplementedwith 10% FCS, 100 μg/ml streptomycin, 100 IU/ml penicillin and 2 mMglutamine. Murine HY Uty-specific T cell clone CTL-10 (Greenfield, A. etal. Nat. Genet. 14, 474-478 (1996), was restimulated weekly with 5×10⁶irradiated male C57BL/6N mouse splenocytes in DMEM supplemented with 10%FCS, 5×10⁻⁵ M 2-mercaptoethanol, 0.56 mM L-arginine, 6 μg/ml folic acid,36 mg/ml L-asparagine, 1 mM sodium pyruvate, 1.4 mM L-glutamine, 0.5mg/ml gentamycin and 40 IU/ml rIL-2.

Engineering of target APC. Full-length cDNA sequences encoding D^(b) andB7.1 were inserted into the same mammalian expression vector pBudCE4.1(Invitrogen), which allowed expression of two heterologous genes througha single transfection. Trypsinized 293T cells (1×10⁷) were resuspendedin serum-free DMEM containing 20 μg of purified pBudCE4.1-D^(b)-B7.1plasmid DNA, incubated on ice for 15 minutes, and electroporated using aBTX ECM830 electroporator (Harvard Apparatus) at 300 V with three 5milli-second pulses. After culture for 3 weeks with 200 μg/ml of zeocin(Invitrogen), zeocin-resistant transfectants were stained withFITC-conjugated D^(b) and phycoerythrin (PE)-conjugated mouse B7-1specific mAbs (eBiosciences), and sorted by a FACSVantage cytometer (BDBiosciences). Stable transfectants expressing D^(b) and B7.1 werefurther selected by limiting dilution and maintained in 100 μg/ml ofzeocin.

cDNA plasmid spotting. To prepare a HY minigene expression DNAconstruct, two synthetic complementary oligonucleotides encoding HYpeptide, Uty₂₄₆₋₂₅₄, MWMHHNMDLI (SEQ ID NO: 1), sense,5′-GGCCGCCATGTGGATGCACCATAATATGGATCTAAT-3′ (SEQ ID NO: 2), andantisense, 5′-CTAGATTAGATCCATATTATGGTGCATCCACATGGC-3′ (SEQ ID NO: 3),were annealed together, and subsequently inserted into the pcDNA3/neovector (Invitrogen, Carlsbad, Calif., USA) using Not I and Xba Irestriction sites. To generate the EGFP-HY minigene construct, sense5′-GATCCCAACACTTAGGTTGGATGCACCATAATATGGATCTAATTTGAT-3′ (SEQ ID NO: 4)and antisense 5′-CTAGATCAAATTAGATCCATATTATGGTGCATCCAACCTAACTGTTGG-3′(SEQ ID NO: 5) oligonucleotides were annealed together, and subsequentlyinserted into the pEGFP-C1 vector (Clontech) using EcoR I and Xba Irestriction sites. After transformation of E. coli DH5a cells, plasmidDNA was purified using an Endofree plasmid preparation kit (Qiagen).

Lipid-DNA complexes. Lipid-DNA complexes were prepared using Effectenetransfection Kit (QIAGEN) by a procedure modified from Ziauddin et al(Ziauddin, J. & Sabatini, D. M. Nature 411, 107-110 (2001)) Briefly,after 3.2 μg DNA was diluted with 30 μl of DNA condensation Buffer ECcontaining 0.4 M sucrose, then 13.5 μl of Enhancer solution was addedand mixed. Following a 5 minute incubation at room temperature, 20 μlEffectene transfection reagent was added and mixed with gentlevortexing. The mixture was at room temperature for 10 minutes, then a ⅓volume of 0.2% gelatin was added. After mixing, 20 μl of the solutionwas transferred into 96-well polypropylene library storage plates (BDBiosciences), for printing on functionalized epoxy microarray slides(TeleChem International), using a manual MicroCaster arrayer equippedwith 8 pins (Schleicher & Schuell Bioscience). Individual pinstransferred a small volume of the ‘lipid-DNA’ solution to the slidewhile touching the slide surface for 200-500 ms. Printed slides werestored at room temperature or 4° C. in a vacuum desiccator.

Reverse transfection. Air-dried DNA-liposome slides were attached to aplastic chamber from the Lab-Tek chambered slide system (Nalge NuncInternational), and placed in a QuadriPerm 4-compartment cell culturedish (Sigma). Healthy 293T cells were harvested by trypsinization andre-suspended in complete DMEM medium at a density of 0.5×10⁶ cells/mland 5 ml of cell suspension was transferred onto the chambered slide.Cells were cultured in a 5% CO₂ humidified incubator at 37° C. for 24-48h. Slides were washed twice with ice-cold PBS, fixed with 3.7%paraformaldehyde in PBS at room temperature for 20 min, and fluorescenceimages were acquired using a Typhoon 8400 scanner (Amersham Pharmacia)or ScanArray Express HT scanner (Perkin Elmer).

Assay of CTL activity on microarray. Antigen specific CTL in completegrowth medium, 3.0×10⁶ cells/ml in 2 mls, were routinely transferredonto the engineered APC monolayer on microarray. Fourteen μl of a150×SR-VAD-FMK (BIOMOL) stock solution was added to the medium. Afterbrief mixing, slides were incubated at 37° C. for 4 hours. Slides werewashed twice with 1× Wash Buffer from the multi-caspase detection kit(BIOMOL), incubated with 3.7% paraformaldehyde in PBS at roomtemperature for 20 min, and washed twice with PBS to remove excessfixative. Fluorescence images were acquired using Typhoon 8400 orScanArray Express HT fluorescence scanners.

Transient transfection. Oligonucleotides encoding HY peptide, MWMHHNMDLI(SEQ ID NO: 1) and QQLGWMHHNMDLI (SEQ ID NO: 6), were synthesized bySigma-Genosys. Influenza A virus nucleoprotein (NP) cDNA in expressionvector was provided by Dr. J. Yewdell (NIH). Engineered 293T cellsstably expressing D^(b) and B7.1 were transiently transfected withplasmid DNA constructs encoding the HY minigene or its EGFP fusion usingEffectene. The APCs transfected with pcDNA3 vector only, pcDNA3-NP, orpEGFP-C1 served as controls.

⁵¹Cr release assay. Following transient transfection with plasmid DNAcontaining HY mini-gene, NP cDNA inserts or vector alone, engineeredAPCs (0.5×10⁶) were labeled with 100 μl ⁵¹Cr at 37° C. for 2 h, washedtwice, dispensed into triplicate cultures at 5×10³ target cells/well in96-well round-bottom microtiter plates (Corning), and incubated for 4 hwith effector T cells (2.5×10⁴ cells/well) in a volume of 200 μl.Radioactivity was determined using a Wallac MicroBetaScintillation/Luminescence Counter (PerkinElmer). Specific lysis wascalculated with the following formula: (experimental release−spontaneousrelease)/(maximal release−spontaneous release)×100. Spontaneous releasewas determined from ⁵¹Cr-labeled target cells incubated in the presenceof medium only, and maximal release in the presence of 5% Triton X-100without CTL.

Flow cytometric cytotoxicity assay. Following transient transfectionwith plasmid DNA containing the HY mini-gene, NP cDNA inserts or vectoralone, 0.5×10⁶ engineered APCs were incubated with CellTracker Orangedye (Molecular Probes) for 30 min at room temperature and recovered for1 h at 37° C. Labeled APCs were then incubated for 3 h with 2.5×10⁶ ofeffector T cells in a volume of 300 μl. Samples were washed twice withPBS, and re-suspended in 300 μl of PBS containing 10 μM SR-VAD-FMK. Cellsuspensions were incubated at 37° C. for another hour, washed twice andanalyzed in a FACSCalibur cytometer with CellQuest software (BDBiosciences).

Example 1 Recombinant Target APCs

Target APCs were engineered from human 293T cells by modifying the 293Tcells to stably express mouse MHC class I molecule, H-2D^(b) to presentan antigen of interest, as well as a costimulatory ligand B7.1. Themouse CTL clone 10 recognized an HY male minor hostocompatabilityantigen peptide, Uty₂₄₆₋₂₅₄ WMHHNMDLI (SEQ ID NO: 7), bound to H-2D^(b).Engineered 293T cells transiently transfected with a mini-gene encodingthe HY peptide epitope were lysed by CTL-10, as detected in ⁵¹Cr-releaseassay and target cell FLICA retention, confirming the specificity ofresponse to the APC and utility of FLICA.

Example 2 Detection of Antigen-Specific CTL Cytotoxicity

Expression of individual cDNAs at defined addresses on microarray wascombined with antigen specific CTL recognition by FLICA retention andlocalization. Vectors containing EGFP, influenza nucleoprotein (NP) HYmini-gene cDNAs, or vector alone, as lipid-DNA complexes in gelatin weredeposited on microarray slides. Gelatin preserved the location ofdeposited lipid-DNA complexes on the microarray. The 293T cellsexpressing mouse H-2 D^(b) and B7.1 were applied to each slide. After 32hours, HY-specific CTL-10 was added, and fluorescence images of theslides were obtained after 4 hours. Intense green fluorescence at GFPcontrol vector spots indicated successful transient transfection of APC(FIG. 2). Importantly, a strong concentration of FLICA was observed,indicated as red fluorescence, where the antigen-encoding HY mini-genewas spotted, but little or no FLICA where the NP or vector alone wasspotted (FIG. 2). These data indicated for the first time that antigenspecific CTL-mediated cytotoxicity was detectable on a reversetransfected microarray.

Example 3 Detection of Antigen from a Processed Polypeptide inRecombinant Target APCs at the Single-Cell Level

A DNA construct encoding the HY peptide, Uty₂₄₂₋₂₅₄ QQLGWMHHNMDLI (SEQID NO: 6) fused to EGFP (GFP-HYmini) was prepared. Expression of theGFP-HY fusion protein was comparable to GFP by reverse transfection onthe microarray (FIG. 3, upper panel). Processing of the GFP-HY fusionprotein to yield the HY peptide by the APC was detected as CTL-inducedconcentration of FLICA (red) where the GFP-HYmini, but not GFP vectorwas spotted (FIG. 3, middle panel). A merged image indicated that asignificant fraction of cells expressing the GFP-HY fusion protein arealso FLICA positive and appear yellow, while such is not the case forGFP-expressing cells (FIG. 3, bottom panel). These results demonstratedthat the CTL microarray assay detected processing and presentation of apeptide epitope generated from a longer protein precursor. In addition,at 5 μm resolution, the observed CTL recognition of APC is likely at thesingle cell level on the microarray, providing a better opportunity toidentify antigens when CTLs are at low frequency.

It is evident from the above results and discussion that the subjectinvention provides an important new means for the detection ofantigen-specific CTLs and for identification of antigens recognized byantigen-specific CTLs. As such, the subject methods and systems find usein a variety of different applications, including research, medical,therapeutic, and other applications. Accordingly, the present inventionrepresents a significant contribution to the field.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method for identifying a cytolytic T lymphocyte(CTL) antigen, the method comprising: contacting a sample comprising acytolytic T lymphocyte (CTL) with an array comprising a plurality ofadherent recombinant antigen-presenting cells (APCs) adhered to asurface of the array at different, discrete locations on the array,wherein the adherent recombinant APCs express different recombinantpolynucleotides encoding different polypeptides, and wherein thedifferent, discrete locations on the array correlate with the differentrecombinant polynucleotides expressed by the adherent recombinant APCs;washing the array; and detecting the presence or absence of caspaseactivity in the plurality of adherent recombinant APCs by detecting thepresence of a fluorescent signal generated from a fluorogenic caspasesubstrate present in said adherent recombinant APCs; wherein thepresence of a fluorescent signal in an adherent recombinant APC isindicative of an antigen-specific interaction between the CTL and theadherent recombinant APC and indicates the recombinant polynucleotide ofthe adherent recombinant APC encodes a polypeptide that comprises a CTLantigen.
 2. The method of claim 1, wherein the recombinantpolynucleotides encode a tumor antigen.
 3. The method of claim 1,wherein the recombinant polynucleotides encode an antigen of anintracellular pathogen.
 4. The method of claim 3, wherein the antigen isa viral antigen, bacterial antigen, antigen of a parasite, or fungalantigen.
 5. The method of claim 1, wherein the recombinantpolynucleotides encode an autoantigen.
 6. The method of claim 1, whereinthe fluorogenic caspase substrate is a fluorogenic multi-caspasesubstrate.
 7. The method of claim 1, wherein the CTL is in a biologicalsample obtained from a subject.
 8. The method of claim 1, wherein theCTL is a CTL clone.
 9. The method of claim 1, wherein said contacting isfor about 1 to 4 hours.
 10. The method of claim 1, wherein saidcontacting is for about 1 hour.
 11. The method of claim 1, wherein theadherent recombinant APCs were produced by plating the APCs onto anarray surface comprising the recombinant polynucleotides deposited on anarray location, wherein said plating is under appropriate conditions foradherence of the APCs to the array surface and introduction of therecombinant polynucleotide into the adherent APC, and wherein the arraylocation at which the recombinant polynucleotide was depositedcorresponds to the location of the adherent APC containing therecombinant polynucleotide.
 12. The method of claim 1, wherein theadherent recombinant APC contains a recombinant polynucleotide encodinga polypeptide fragment.
 13. The method of claim 12, wherein thepolypeptide fragment provides a T cell antigen that is presented inClass I MHC for recognition by a CTL specific for that antigen.